Pharmacological modulation of autophagy enhances Newcastle disease virus-mediated oncolysis in drug-resistant lung cancer cells
- Ke Jiang†1,
- Yingchun Li†2,
- Qiumin Zhu3,
- Jiansheng Xu4,
- Yupeng Wang1,
- Wuguo Deng1,
- Quentin Liu1,
- Guirong Zhang2Email author and
- Songshu Meng1Email author
© Jiang et al.; licensee BioMed Central Ltd. 2014
Received: 27 January 2014
Accepted: 22 July 2014
Published: 30 July 2014
Oncolytic viruses represent a promising therapy against cancers with acquired drug resistance. However, low efficacy limits its clinical application. The objective of this study is to investigate whether pharmacologically modulating autophagy could enhance oncolytic Newcastle disease virus (NDV) strain NDV/FMW virotherapy of drug-resistant lung cancer cells.
The effect of NDV/FMW infection on autophagy machinery in A549 lung cancer cell lines resistant to cisplatin (A549/DDP) or paclitaxel (A549/PTX) was investigated by detection of GFP-microtubule-associated protein 1 light chain 3 (GFP-LC3) puncta, formation of double-membrane vesicles and conversion of the nonlipidated form of LC3 (LC3-I) to the phosphatidylethanolamine-conjugated form (LC3-II). The effects of autophagy inhibitor chloroquine (CQ) and autophagy inducer rapamycin on NDV/FMW-mediated antitumor activity were evaluated both in culture cells and in mice bearing drug-resistant lung cancer cells.
We show that NDV/FMW triggers autophagy in A549/PTX cells via dampening the class I PI3K/Akt/mTOR/p70S6K pathway, which inhibits autophagy. On the contrary, NDV/FMW infection attenuates the autophagic process in A549/DDP cells through the activation of the negative regulatory pathway. Furthermore, combination with CQ or knockdown of ATG5 significantly enhances NDV/FMW-mediated antitumor effects on A549/DDP cells, while the oncolytic efficacy of NDV/FMW in A549/PTX cells is significantly improved by rapamycin. Interestingly, autophagy modulation does not increase virus progeny in these drug resistant cells. Importantly, CQ or rapamycin significantly potentiates NDV/FMW oncolytic activity in mice bearing A549/DDP or A549/PTX cells respectively.
These results demonstrate that combination treatment with autophagy modulators is an effective strategy to augment the therapeutic activity of NDV/FMW against drug-resistant lung cancers.
KeywordsNewcastle disease virus Autophagy Apoptosis Drug resistance Lung cancer Virotherapy
Acquired drug resistance to first-line chemotherapeutics, such as cisplatin and paclitaxel, is a major factor contributing to chemotherapy failure in non-small cell lung cancer (NSCLC) patients [1, 2]. Oncolytic viruses (OVs) are emerging as new cancer therapeutic approaches with great potential for the treatment of drug-resistant lung cancers . We previously reported that the oncolytic Newcastle disease virus (NDV) induces apoptosis in cisplatin-resistant A549 (A549/DDP) cells in vitro and in vivo. NDV is an avian paramyxovirus that selectively replicates in a variety of tumor cells but not in normal human cells . NDV strains such as LaSota, Ulster , 73-T , NDV/FMW [8, 9], and NDV- HUJ [10, 11] have displayed oncolytic effects in lung cancer cells. Notably, in addition to triggering apoptosis in chemo-resistant malignant primary melanoma , oncolytic NDV induces efficient oncolysis in human lung adenocarcinoma A549 cells over-expressing Bcl-xL, a known anti-apoptotic protein . These studies and studies from our lab indicate a potential role of oncolytic NDV in the treatment of drug-resistant lung cancers. However, it remains a challenge to improve the efficacy of NDV in drug-resistant NSCLC cells in preclinical and clinical tests.
Oncolytic NDV is known to trigger apoptosis pathways in infected tumor cells [4, 8, 10, 14–16]. In addition to targeting the cellular apoptosis machinery, we recently reported that oncolytic NDV induces autophagy in U251 human glioma cells to promote virus production , suggesting that autophagy may be involved in NDV-induced oncolysis. Autophagy is a conserved homeostatic mechanism of lysosomal degradation . The hallmark of autophagy is a double-membraned autophagosome that engulfs long-lived cytoplasmic macromolecules and damaged organelles . Autophagy is mainly modulated by the mTOR (mammalian target of rapamycin) and PI3K (phosphatidylinositol 3-kinase) pathways, which are class I (inhibitory to autophagy) and class III (necessary for the execution of autophagy) modulators [20, 21]. Accumulating evidence reveals that OVs interact with the autophagy machinery in infected tumor cells, and autophagy plays a role in OV-mediated cancer cell death [22–24]. Of note, a number of studies reported that the pharmacological modulation of autophagy augments the anti-tumor effects of OVs, such as the oncolytic adenovirus OBP-405 in combination with the autophagy inducers temozolomide, rapamycin and RAD001 in glioma cells , dl922-947 in combination with the autophagy inhibitor chloroquine (CQ) in glioma cells , Ad-cycE with rapamycin in lung cancer cells . In addition, autophagy plays critical roles in both innate and adaptive immuninity. It has been shown that autophagy enhances tumor immunogenicity via releasing damage-associated molecular pattern (DAMP) molecules by dying cells with autophagy and promoting antigen cross presentation from cancer cells by DCs to naive T cells [28, 29]. Since OV infections can interact with the cellular autophagy machinery, OV in combination with an autophagy modulator would enhance the antitumor immune responses, thereby improving OV-mediated efficacy [29–31]. Together, data from these studies strongly indicate that targeting autophagy may be utilized as a novel strategy for enhancing the oncolytic virotherapy of cancers.
The objective of this study was to investigate whether pharmacologically targeting autophagy could enhance NDV virotherapy in drug-resistant lung cancer cells. We first dissected the interaction between NDV and the cellular autophagy machinery in cisplatin- and paclitaxel-resistant A549 lung cancer cells and further demonstrated that the modulation of autophagy with rapamycin or CQ enhances the NDV-mediated anti-tumor effects on drug-resistant A549 cells in vitro and in vivo. Therefore, our results suggest that combination with chemotherapeutic agents that modulate autophagy may be a potential strategy to optimize the clinical efficacy of oncolytic NDV.
Cell lines, mice and virus preparation
A549 human lung cancer cell line and chicken embryo fibroblast cell line DF1 was purchased from American Type Culture Collection (ATCC) and cultured at 37°C and 5% CO2 in DMEM supplemented with 10% fetal bovine serum (FBS). Cisplatin-resistant A549 (A549/DDP) cells  were cultured in DMEM containing 2 μg/mL cisplatin (Sigma) to maintain resistance. An A549-derived paclitaxel-resistant sub-line, A549/PTX, was kindly provided by Dr. Sang Kook Lee (Seoul National University) and cultured in RPMI 1640 containing 100 ng/mL paclitaxel (Sigma) to maintain resistance. The cells were cultured in complete media without cisplatin or paclitaxel for 3 days before performing experiments. The NDV strain NDV/FMW, which has been previously shown to be oncolytic in A549/DDP and parental cells [4, 8], was used throughout the study. Virus passaging, propagation, and titration were performed as previously described, and virus titer was expressed as log10 50% tissue culture infective dose (TCID50) . BALB/c nude mice (female, 4–6 weeks old) were purchased from the Experimental Animal Center of Dalian Medical University (Dalian, China) and all procedures involving animals and their care complied with the China National Institutes of Healthy Guidelines for the Care and Use of Laboratory Animals. Ethical approval for the study was granted by the Ethics Committee of Dalian Medical University.
Antibodies and reagents
The monoclonal anti-Beclin-1 antibody and high-mobility group box1(HMGB1) were purchased from Santa Cruz. The polyclonal rabbit anti-microtubule-associated protein 1A/1B-light chain 3 (LC3) and a monoclonal antibody against β-Actin were obtained from Sigma. The following antibodies were purchased from Cell Signaling Technology: cleaved caspase-3 and phospho-specific antibodies to mTOR (Ser2448), Akt (Ser473) and p70 ribosomal protein S6 kinase (S6K) (Thr389), along with total antibodies directed against mTOR, Akt, and p70S6K. Rapamycin and chloroquine (CQ) were purchased from Sigma.
A549/DDP, A549/PTX, and parental A549 cells were infected with NDV/FMW at a multiplicity of infection (MOI) of 10, or they were sham-infected with phosphate-buffered saline (PBS), at 37°C for 1 h in serum-free DMEM. The cells were washed three times with PBS and incubated at 37°C in reduced serum (1% FBS)-containing media. For the pharmacological modulation of autophagy, cells were treated with rapamycin (100 nM) or CQ (5 μM) for 30 min prior to virus infection. Subsequently, the cells were infected with NDV/FMW in the presence or absence of various compounds for 1 h and then cultured in fresh DMEM or RPMI 1640 containing rapamycin or CQ for the indicated times. For experiments that involved the determination of virus yield, tumor cells were infected with NDV/FMW at an MOI of 0.01, and multi-step viral growth curves were measured as previously described .
Cell transfection and fluorescence microscopy
Tumor cells were transfected with a plasmid expressing green fluorescent protein (GFP)-LC3 using Lipofectamine 2000 according to the manufacturer’s instructions. Dot formation by GFP-LC3 was detected with a fluorescence microscope (BX50, Olympus) following drug treatment and/or NDV/FMW infection. Transfected cells with five or more puncta were considered to have accumulated autophagosomes. A total of 100 transfected cells were examined per well in triplicate from three independent experiments.
RNA interference was used to knock down ATG5, a key gene for autophage formation. Two siRNA oligonucleotides were used: ① 5'-TGA TAT AGC GTG AAA CAA G-3' ; ② 5'-CAA CTT GTT TCA CGC TAT A--3' . Transfection of siRNA was performed as described previously [17, 35]. A scrambled siRNA was used as a negative control. The silencing efficiency was detected by immunoblot. At 48 h after transfection, cells were infected with NDV/FMW at an MOI of 10 for various times.
Transmission electron microscopy analysis
Standard transmission electron microscopy (TEM) was performed as previously described . Briefly, 24 h after NDV/FMW infection, the cells were fixed and embedded. Thin sections (90 nm) were examined at 80 kV with a JEOL 1200EX transmission electron microscope. Approximately 15 cells were counted, and autophagosomes were defined as double-membrane vacuoles measuring 0.5 or 2.0 μm.
Cell proliferation assay
Tumor cells were seeded into 96-well plates, and cell growth was measured daily by the MTT assay as previously described . The experiments were repeated three times.
Flow cytometric analysis of apoptosis
Apoptosis was quantified using flow cytometry as previously described . Briefly, tumor cells were seeded at 1 × 105 cells/dish in 60-mm dishes and treated with NDV/FMW at an MOI of 10. Floating cells and cell pellets were prepared for the annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) double-staining procedure. The cell population in the lower right quadrant (PI-negative, annexin V-positive) corresponds to apoptotic cells. The data was determined in three independent experiments.
Immunoblot (IB) assays were performed as described previously . Densitometry analysis of the specific protein expression was performed using a calibrated GS-670 densitometer. All IB experiments were performed in duplicate.
Nude mice were subcutaneously inoculated in the flank with A549/DDP and A549/PTX cells (5 × 106 cells in 100 μL PBS/mouse) to induce tumor development. When tumors reached an average volume of 200 mm3, tumor-bearing mice were intratumorally inoculated with NDV/FMW. Mice were randomly divided into four groups (six mice per group): (a) vehicle treatment, (b) intraperitoneal (i.p.) treatment with rapamycin (5 mg/kg) or CQ (45 mg/kg) alone three times a week, (c) intratumoral administration with NDV/FMW (1 × 107 TCID50 per dose) three times a week, and (d) NDV/FMW treatment in combination with CQ or rapamycin (same dose as described previously) administered 1 d prior to virus injection. One week after treatment, two mice (of six) were sacrificed, and tumor sections (5 μm) were subjected to either hematoxylin–eosin (H&E) staining or terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay as previously described [4, 9]. TUNEL-positive (brown staining) cells were characterized as apoptotic cells, and 10 randomly selected microscopic fields in each group were examined to calculate the ratio of TUNEL-positive cells. Tumor tissue samples from two different mice (of six) from each treatment group were subjected to immunoblot analysis to evaluate cleaved caspase-3 levels or LC3II abundance. Excised tumors from the other two animals (of six) were subjected to virus isolation.
For the in vivo oncolysis study, 10 mice were included in each treatment group, and the four mouse groups were treated as described above for two weeks. At five-day intervals, mice were examined for tumor growth or survival. Tumor diameter was measured with a caliper, and tumor volume was calculated based on the following formula: volume = (greatest diameter) × (smallest diameter) 2/2. The experiment was terminated when tumors reached 1 cm3 in volume and/or symptomatic tumor ulceration occurred, and the surviving mice were sacrificed under anesthesia.
Comparisons of data for all groups in the viral propagation and cytotoxicity assays were first performed using one-way analysis of variance (ANOVA). Multiple comparisons between treatment groups and controls were evaluated using Dunnett’s least significant difference (LSD) test. To assess in vivo oncolytic effects, statistical significance between groups was calculated using the LSD test in SPSS 17.0 software (SPSS Inc., Chicago, IL, USA). A p < 0.05 was considered statistically significant.
NDV/FMW induces autophagosome formation in paclitaxel-resistant A549 cells but attenuates the autophagic process in cisplatin-resistant A549 cells.
To determine whether NDV/FMW perturbs autophagosome formation in drug-resistant A549 cells, we detected GFP-LC3 dot formation, which is generally regarded as an autophagosome . A549/DDP, A549/PTX, and parental cells were transfected with GFP-LC3 and then mock-infected or infected with NDV/FMW at an MOI of 10. As shown in Figures 1B and D, the GFP-LC3 redistribution into discrete dots was significantly increased in NDV/FMW -infected A549/PTX (**p < 0.01) and parental (**p < 0.01) cells at 24 hpi, while a diffuse cytoplasmic distribution of fluorescence was observed in mock-treated A549/PTX and parental cells. Interestingly, marked punctated GFP-LC3 accumulation was observed in mock-infected A549/DDP cells (Figure 1C), suggesting a high basal level of autophagy. However, upon NDV/FMW infection, the number of A549/DDP cells with punctated GFP-LC3 was significantly diminished compared to basal levels (Figure 1C, **p < 0.01). Control cells treated with the autophagy inducer rapamycin exhibited typical GFP-LC3 dot formation. In addition, TEM-based ultrastructural analysis of the formation of double-membrane vesicles (autophagosomes) confirmed the above findings (Figures 1E, F, and G). Therefore, these results indicate that NDV/FMW induces autophagosome formation in A549/PTX and parental cells, whereas it inhibits the autophagic process in A549/DDP cells.
NDV/FMW infection perturbs autophagic signaling pathways in drug-resistant A549 cells
Beclin-1 forms a complex with class III PI3K and plays an essential role in controlling the first steps of autophagy commitment . We found that beclin-1 expression was up-regulated in a time-dependent manner in NDV-infected A549/PTX and parental cells (Figure 2B, left and right panels), suggesting that beclin-1 may participate in the induction of autophagosome formation in these cells during NDV/FMW infection. Upon NDV/FMW infection, the expression of beclin-1 in A549/DDP cells was nearly unchanged from 4 to 12 hpi and was completely diminished at 24 hpi (Figure 2B, middle panel), suggesting that NDV/FMW infection decreases beclin-1 expression in the late stage of infection. Therefore, these data indicate that the class III PI3K/Beclin-1 pathway may be involved in the interplay between NDV/FMW and the cellular autophagy machinery in drug-resistant A549 and parental cells.
Pharmacological modulation of autophagy enhances NDV/FMW-induced cytotoxicity
Knockdown of autophagy-related gene ATG5augments NDV/FMW-mediated oncolysis in cisplatin-resistant A549 cells
The execution of cell death requires an orchestrated interplay between three important processes: apoptosis, necrosis and autophagy [48, 49]. Data in Figure 3C indicated that dying cells that are double positive for PI and annexin were detected in A549/DDP cells treated with NVD/FMW or NVD/FMW with CQ at 48 hpi, suggesting that some of the cells might die via necrosis or a late necrosis consecutive to apoptosis upon virus infection and the combination treatment. To explore whether NDV-induced necrosis was modulated by regulation of autophagy, we knocked down the ATG5 protein expression using specific siRNA targeting ATG5 in A549/DDP cells. As shown in Figure 4D, at 48 hpi, markedly more dying cells that are double positive for PI and annexin were observed in ATG5-deficient A549/DDP cells than in A549/DDP cells transfected with control siRNA, suggesting that modulation of autophagy may exert an effect on NDV/FMW-induced apoptosis and necrosis. Consistent with the FACS data, we observed enhanced releasing of HMGB1 protein, a known marker of immunogenic cell death at late stages , in ATG5-deficient A549/DDP cells at 48 hpi compared to A549/DDP cells transfected with control siRNA (Figure 4C). We did not observe marked increase in dying cells that are double positive for PI and annexin as well as releasing of HMGB1 in ATG5-deficient A549/PTX cells upon NDV infection (data not shown). Collectively, these results suggested that ATG5 knockdown enhanced NDV/FMW-mediated oncolysis in A549/DDP cells.
Autophagy modulation does not increase virus progeny in drug resistant cells
CQ or rapamycin potentiates NDV/FMW oncolytic activity in mice bearing drug-resistant lung cancer cells
We further investigated whether the in vivo combination treatments resulted in enhanced inhibition of tumor cell growth as demonstrated in our in vitro experiments. The treatment of tumors bearing A549/PTX cells with rapamycin alone or the addition of CQ to mice bearing A549/DDP-derived tumors had negligible therapeutic effects on tumor growth (Figures 6E and F). As expected, NDV/FMW virotherapy markedly reduced tumor growth compared with vehicle treatment (Figures 6E and F, p < 0.05, respectively). Interestingly, the combination of NDV/FMW with rapamycin induced a significant reduction in tumor volume 5 days earlier than virus alone (Figure 6E, *p < 0.05; **p < 0.01), and combination therapy also resulted in significant tumor growth inhibition compared with virus alone (Figure 6E). Similar effects were detected in mice bearing A549/DDP cells treated with NDV in combination with CQ (Figure 6F). The difference in tumor volume assessed at each time point became statistically significant at day 35 (all p values were lower than 0.05) and day 45 (all p values were lower than 0.01). Together, these data indicate that CQ and rapamycin are effective in increasing the antitumor activity of NDV/FMW in cisplatin- and paclitaxel-resistant A549 lung cancer cell mouse models.
Currently, the major limitation in the development of OVs in clinical trials is the low efficacy of the viruses in vivo. Here, we provide in vitro and in vivo evidence that pretreatment with the autophagy inhibitor CQ enhances NDV/FMW-mediated antitumor effects in A549/DDP cells via the inhibition of autophagy, while the autophagy inducer rapamycin improves the oncolytic efficacy of NDV/FMW in A549/PTX cells through enhanced autophagy, suggesting that the combined administration of autophagy modulators with oncolytic NDV/FMW may improve virotherapy in lung cancer cells resistant to various chemotherapies.
It is well known that viral infection and the cellular autophagy machinery have complex interconnections. We previously observed that NDV induces autophagy in U251 glioma cells . In the current study, we showed that oncolytic NDV/FMW triggers autophagy in A549/PTX cells via the inhibition of the class I PI3K/Akt/mTOR/p70S6K pathway, which negatively regulates autophagy. However, NDV/FMW infection blocks the autophagic process in A549/DDP cells through the activation of the negative regulatory pathway. A plausible explanation for the diverse strategies that regulate the cellular autophagy machinery utilized by NDV/FMW is that the induction or inhibition of autophagy by NDV/FMW may depend on the role of autophagy in these drug-resistant lung cancer cells. Autophagy may act as a survival or cell death mechanism in drug-resistant cancers. A previous study reported that cisplatin treatment induces autophagy in A549 cells, and the acquired cisplatin resistance in A549 cells is associated with enhanced autophagy . Another study suggested that cisplatin-induced autophagy might provide a prosurvival role in cisplatin-resistant SKOV3 ovarian cancer cells . In line with these findings, we also detected high basal levels of autophagy in A549/DDP cells, suggesting that autophagy may act as a survival mechanism in cisplatin-resistant A549 lung cancer cells. Therefore, it is reasonable that, to induce oncolysis, oncolytic NDV/FMW should inhibit the autophagic process in A549/DDP cells. Surprisingly, the role of autophagy in paclitaxel-resistant cancer cells remains controversial. Ajabnoor et al. reported that an increased autophagic response was observed in paclitaxel-resistant MCF-7 breast cancer cells with reduced phosphor-mTOR and a relative resistance to the mTOR inhibitors rapamycin and RAD001 , suggesting that autophagy may act as a survival mechanism in paclitaxel-resistant breast cancer cells. However, Veldhoen et al. demonstrated that paclitaxel inhibits autophagy in MCF-7 and SK-BR-3 breast cancer cells . Moreover, Veldhoen et al. showed that primary breast tumors that express diminished levels of autophagy-initiating genes were resistant to taxane therapy , suggesting that autophagy may act as a cell death mechanism in PTX-resistant breast cancer cells. In this study, we did not observe an increased basal level of autophagy in A549/PTX cells compared with parental A549 cells, indicating that autophagy may not act as a survival mechanism in A549/PTX cells. Accordingly, NDV/FMW infection resulted in the induction of autophagy in A549/PTX cells, suggesting that autophagy may play a positive role for NDV/FMW to exert its oncolytic effect in these cells. Interestingly, although NDV/FMW triggered autophagy in A549 cells, combination treatment with either rapamycin or CQ did not induce increased apoptosis, indicating that autophagy may not be involved in NDV/FMW-induced oncolysis in A549 cells.
Currently, pharmacological autophagy modulators such as rapamycin and CQ and their analogs or derivatives have been widely used in combination with OVs to enhance virotherapy for a variety of cancers in preclinical trials [26, 40–42]. However, whether autophagy inducers or inhibitors are used in combination virotherapy may be virus strain- and cancer line-dependent. Here, we presented in vitro evidence that rapamycin enhances NDV/FMW-mediated oncolysis in A549/PTX cells via increased autophagy, while CQ augments the antitumor effects of NDV/FMW on A549/DDP cells via inhibition of autophagosome-lysosome fusion, which is in agreement with the way in which NDV/FMW perturbs the cellular autophagy machinery in these drug-resistant lung cancer cells. The increase in NDV/FMW-mediated cytotoxicity in the presence of autophagy modulators may be due to the augmented activation of apoptosis and necrosis as demonstrated by enhanced capase-3 activation and increased numbers of apoptotic and necrotic cells. However, combination treatments may exert their effects via enhanced viral propagation. Interestingly, CQ treatment significantly reduced the yield of NDV/FMW progeny in A549/DDP cells, while pretreatment with rapamycin did not alter viral titers, suggesting that the increase in viral cytotoxicity in the presence of the autophagy inhibitor CQ might be due to enhanced activation of apoptosis and necrosis rather than altered viral replication. In contrast, pretreatment with either rapamycin or CQ did not significantly alter virus yield in NDV/FMW-infected A549/PTX cells, excluding a role for viral propagation in enhanced viral cytotoxicity by combination treatments. This notion is in agreement with studies of other OVs with combination therapies. Botta et al. reported that the inhibition of autophagy by CQ enhances the effects of the oncolytic adenovirus dl922-947 against glioma cells, while viral replication is not increased by autophagy modulation . Our in vivo data further indicated that NDV/FMW in combination with rapamycin or CQ induces enhanced caspase-3 activation accompanied by increased LC3II abundance in A549/PTX- and A549/DDP-derived tumors. However, it should be noted that the contribution of autophagic modulators rapamycin and CQ on antitumor immunity and thus the efficacy of oncolytic virotherapy have not been taken into consideration in our in vivo experiments. OVs can induce autophagy and immunogenic cancer cell death which may be potentiated by co-administration with autophagy modulators as modulation of autophagy may enhance tumor immunogenecity . It was reported that the combination of oncolytic adenovirus with low-dose temozolomide increased tumor cell autophagy, elicited antitumor immune responses in chemotherapy-refractory cancer patients . Therefore, further in vivo studies are required to clarify the roles of rapamycin and CQ in antitumor immunity contributing to the overall efficacy of NDV-mediated virotherapy.
In the current study, we provide evidence that pharmacological autophagy modulation enhanced the in vitro and in vivo oncolytic effects of NDV/FMW in drug-resistant lung cancer cells. Our findings suggest that the combination of NDV/FMW and autophagy modulators may be a novel treatment option for lung cancer patients with recurrent disease after cisplatin- or paclitaxel-based first-line chemotherapy. Of note, recent study demonstrated that drug-resistant NSCLC cell lines may display a stem-like signature [55, 56], linking cancer stem cell with drug resistance. Therefore, it will be interesting to extend our study to lung cancer stem cell.
Microtubule-associated protein 1 light chain 3
Multiplicity of infection
Autophagy-related gene 5
Mammalian target of rapamycin
3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide
Newcastle disease virus
Non-small-cell lung carcinoma
This work was supported by the National Science Foundation of China (81372471). This work was also supported by grants from the Program for Changjiang Scholars and Innovative Research Teams in University (02738960345 k) and the Priority Academic Program Development of Jiangsu Higher Education Institutions. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
- Brozovic A, Osmak M: Activation of mitogen-activated protein kinases by cisplatin and their role in cisplatin-resistance. Cancer Lett. 2007, 251 (1): 1-16.View ArticlePubMed
- Hsu DS, Balakumaran BS, Acharya CR, Vlahovic V, Walters KS, Garman K, Anders C, Riedel RF, Lancaster J, Harpole D, Dressman HK, Nevins JR, Febbo PG, Potti A: Pharmacogenomic strategies provide a rational approach to the treatment of cisplatin-resistant patients with advanced cancer. J Clin Oncol. 2007, 25 (28): 4350-4357.View ArticlePubMed
- Beljanski V, Hiscott J: The use of oncolytic viruses to overcome lung cancer drug resistance. Curr Opin Virol. 2012, 2 (5): 629-635.View ArticlePubMed
- Meng S, Zhou Z, Chen F, Kong X, Liu H, Jiang K, Liu W, Hu M, Zhang X, Ding C, Wu Y: Newcastle disease virus induces apoptosis in cisplatin-resistant human lung adenocarcinoma A549 cells in vitro and in vivo. Cancer Lett. 2012, 317 (1): 56-64.View ArticlePubMed
- Reichard KW, Lorence RM, Cascino CJ, Peeples ME, Walter RJ, Fernando MB, Reyes HM, Greager JA: Newcastle disease virus selectively kills human tumor cells. J Surg Res. 1992, 52 (5): 448-453.View ArticlePubMed
- Schirrmacher V, Bai L, Umansky V, Yu L, Xing Y, Qian Z: Newcastle disease virus activates macrophages for anti-tumor activity. Int J Oncol. 2000, 16 (2): 363-373.PubMed
- Phuangsab A, Lorence RM, Reichard KW, Peeples ME, Walter RJ: Newcastle disease virus therapy of human tumor xenografts: antitumor effects of local or systemic administration. Cancer Lett. 2001, 172 (1): 27-36.View ArticlePubMed
- Bian J, Wang K, Kong X, Liu H, Chen F, Hu M, Zhang X, Jiao X, Ge B, Wu Y, Meng S: Caspase- and p38-MAPK-dependent induction of apoptosis in A549 lung cancer cells by Newcastle disease virus. Arch Virol. 2011, 156 (8): 1335-1344.View ArticlePubMed
- Wu Y, Zhang X, Wang X, Wang L, Hu S, Liu X, Meng S: Apoptin enhances the oncolytic properties of Newcastle disease virus. Intervirology. 2012, 55 (4): 276-286.View ArticlePubMed
- Yaacov B, Eliahoo E, Lazar I, Ben-Shlomo M, Greenbaum I, Panet A, Zakay-Rones Z: Selective oncolytic effect of an attenuated Newcastle disease virus (NDV-HUJ) in lung tumors. Cancer Gene Ther. 2008, 15 (12): 795-807.View ArticlePubMed
- Yaacov B, Lazar I, Tayeb S, Frank S, Izhar U, Lotem M, Perlman R, Ben-Yehuda D, Zakay-Rones Z, Panet A: Extracellular matrix constituents interfere with Newcastle disease virus spread in solid tissue and diminish its potential oncolytic activity. J Gen Virol. 2012, 93 (Pt 8): 1664-1672.View ArticlePubMed
- Lazar I, Yaacov B, Shiloach T, Eliahoo E, Kadouri L, Lotem M, Perlman R, Zakay-Rones Z, Panet A, Ben-Yehuda D: The oncolytic activity of Newcastle disease virus NDV-HUJ on chemoresistant primary melanoma cells is dependent on the proapoptotic activity of the inhibitor of apoptosis protein Livin. J Virol. 2010, 84 (1): 639-646.PubMed CentralView ArticlePubMed
- Mansour M, Palese P, Zamarin D: Oncolytic specificity of Newcastle disease virus is mediated by selectivity for apoptosis-resistant cells. J Virol. 2011, 85 (12): 6015-6023.PubMed CentralView ArticlePubMed
- Szeberenyi J, Fabian Z, Torocsik B, Kiss K, Csatary LK: Newcastle disease virus-induced apoptosis in PC12 pheochromocytoma cells. Am J Ther. 2003, 10 (4): 282-288.View ArticlePubMed
- Elankumaran S, Rockemann D, Samal SK: Newcastle disease virus exerts oncolysis by both intrinsic and extrinsic caspase-dependent pathways of cell death. J Virol. 2006, 80 (15): 7522-7534.PubMed CentralView ArticlePubMed
- Fabian Z, Csatary CM, Szeberenyi J, Csatary LK: p53-independent endoplasmic reticulum stress-mediated cytotoxicity of a Newcastle disease virus strain in tumor cell lines. J Virol. 2007, 81 (6): 2817-2830.PubMed CentralView ArticlePubMed
- Meng C, Zhou Z, Jiang K, Yu S, Jia L, Wu Y, Liu Y, Meng S, Ding C: Newcastle disease virus triggers autophagy in U251 glioma cells to enhance virus replication. Arch Virol. 2012, 157 (6): 1011-1018.View ArticlePubMed
- Xie Z, Klionsky DJ: Autophagosome formation: core machinery and adaptations. Nat Cell Biol. 2007, 9 (10): 1102-1109.View ArticlePubMed
- Kraft C, Martens S: Mechanisms and regulation of autophagosome formation. Curr Opin Cell Biol. 2012, 24 (4): 496-501.View ArticlePubMed
- Kroemer G, Marino G, Levine B: Autophagy and the integrated stress response. Mol Cell. 2010, 40 (2): 280-293.PubMed CentralView ArticlePubMed
- Glick D, Barth S, Macleod KF: Autophagy: cellular and molecular mechanisms. J Pathol. 2010, 221 (1): 3-12.PubMed CentralView ArticlePubMed
- Meng S, Xu J, Wu Y, Ding C: Targeting autophagy to enhance oncolytic virus-based cancer therapy. Expert Opin Biol Ther. 2013, 13 (6): 863-873.View ArticlePubMed
- Rodriguez-Rocha H, Gomez-Gutierrez JG, Garcia-Garcia A, Rao XM, Chen L, McMasters KM, Zhou HS: Adenoviruses induce autophagy to promote virus replication and oncolysis. Virology. 2011, 416 (1–2): 9-15.PubMed CentralView ArticlePubMed
- Jiang H, White EJ, Rios-Vicil CI, Xu J, Gomez-Manzano C, Fueyo J: Human adenovirus type 5 induces cell lysis through autophagy and autophagy-triggered caspase activity. J Virol. 2011, 85 (10): 4720-4729.PubMed CentralView ArticlePubMed
- Yokoyama T, Iwado E, Kondo Y, Aoki H, Hayashi Y, Georgescu MM, Sawaya R, Hess KR, Mills GB, Kawamura H, Hashimoto Y, Urata Y, Fujiwara T, Kondo S: Autophagy-inducing agents augment the antitumor effect of telerase-selve oncolytic adenovirus OBP-405 on glioblastoma cells. Gene Ther. 2008, 15 (17): 1233-1239.View ArticlePubMed
- Botta G, Passaro C, Libertini S, Abagnale A, Barbato S, Maione AS, Hallden G, Beguinot F, Formisano P, Portella G: Inhibition of autophagy enhances the effects of E1A-defective oncolytic adenovirus dl922-947 against glioma cells in vitro and in vivo. Hum Gene Ther. 2012, 23 (6): 623-634.View ArticlePubMed
- Cheng PH, Lian S, Zhao R, Rao XM, McMasters KM, Zhou HS: Combination of autophagy inducer rapamycin and oncolytic adenovirus improves antitumor effect in cancer cells. Virol J. 2013, 10: 293-PubMed CentralView ArticlePubMed
- Bartlett DL, Liu Z, Sathaiah M, Ravindranathan R, Guo Z, He Y, Guo ZS: Oncolytic viruses as therapeutic cancer vaccines. Mol Cancer. 2013, 12 (1): 103-PubMed CentralView ArticlePubMed
- Guo ZS, Liu Z, Bartlett DL: Oncolytic Immunotherapy: Dying the Right Way is a Key to Eliciting Potent Antitumor Immunity. Front Oncol. 2014, 4: 74-PubMed CentralPubMed
- Liikanen I, Ahtiainen L, Hirvinen ML, Bramante S, Cerullo V, Nokisalmi P, Hemminki O, Diaconu I, Pesonen S, Koski A, Kangasniemi L, Pesonen SK, Oksanen M, Laasonen L, Partanen K, Joensuu T, Zhao F, Kanerva A, Hemminki A: Oncolytic adenovirus with temozolomide induces autophagy and antitumor immune responses in cancer patients. Mol Ther. 2013, 21 (6): 1212-1223.PubMed CentralView ArticlePubMed
- Jiang ZK, Johnson M, Moughon DL, Kuo J, Sato M, Wu L: Rapamycin enhances adenovirus-mediated cancer imaging and therapy in pre-immunized murine hosts. PLoS One. 2013, 8 (9): e73650-PubMed CentralView ArticlePubMed
- Kim EH, Min HY, Chung HJ, Song J, Park HJ, Kim S, Lee SK: Anti-proliferative activity and suppression of P-glycoprotein by (−)-antofine, a natural phenanthroindolizidine alkaloid, in paclitaxel-resistant human lung cancer cells. Food Chem Toxicol. 2012, 50 (3–4): 1060-1065.View ArticlePubMed
- Zou Z, Yuan Z, Zhang Q, Long Z, Chen J, Tang Z, Zhu Y, Chen S, Xu J, Yan M, Wang J, Liu Q: Aurora kinase A inhibition-induced autophagy triggers drug resistance in breast cancer cells. Autophagy. 2012, 8 (12): 1798-1810.PubMed CentralView ArticlePubMed
- Shingu T, Chumbalkar VC, Gwak HS, Fujiwara K, Kondo S, Farrell NP, Bogler O: The polynuclear platinum BBR3610 induces G2/M arrest and autophagy early and apoptosis late in glioma cells. Neuro Oncol. 2010, 12 (12): 1269-1277.PubMed CentralPubMed
- Sun Y, Yu S, Ding N, Meng C, Meng S, Zhang S, Zhan Y, Qiu X, Tan L, Chen H, Song C, Ding C: Autophagy benefits the replication of Newcastle disease virus in chicken cells and tissues. J Virol. 2014, 88 (1): 525-537.PubMed CentralView ArticlePubMed
- Chen L, Meng S, Wang H, Bali P, Bai W, Li B, Atadja P, Bhalla KN, Wu J: Chemical ablation of androgen receptor in prostate cancer cells by the histone deacetylase inhibitor LAQ824. Mol Cancer Ther. 2005, 4 (9): 1311-1319.View ArticlePubMed
- Kabeya Y, Mizushima N, Ueno T, Yamamoto A, Kirisako T, Noda T, Kominami E, Ohsumi Y, Yoshimori T: LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J. 2000, 19 (21): 5720-5728.PubMed CentralView ArticlePubMed
- Ren JH, He WS, Nong L, Zhu QY, Hu K, Zhang RG, Huang LL, Zhu F, Wu G: Acquired cisplatin resistance in human lung adenocarcinoma cells is associated with enhanced autophagy. Cancer Biother Radiopharm. 2010, 25 (1): 75-80.View ArticlePubMed
- Yue Z, Jin S, Yang C, Levine AJ, Heintz N: Beclin 1, an autophagy gene essential for early embryonic development, is a haploinsufficient tumor suppressor. Proc Natl Acad Sci U S A. 2003, 100 (25): 15077-15082.PubMed CentralView ArticlePubMed
- Alonso MM, Jiang H, Yokoyama T, Xu J, Bekele NB, Lang FF, Kondo S, Gomez-Manzano C, Fueyo J: Delta-24-RGD in combination with RAD001 induces enhanced anti-glioma effect via autophagic cell death. Mol Ther. 2008, 16 (3): 487-493.View ArticlePubMed
- Lun XQ, Jang JH, Tang N, Deng H, Head R, Bell JC, Stojdl DF, Nutt CL, Senger DL, Forsyth PA, McCart JA: Efficacy of systemically administered oncolytic vaccinia virotherapy for malignant gliomas is enhanced by combination therapy with rapamycin or cyclophosphamide. Clin Cancer Res. 2009, 15 (8): 2777-2788.View ArticlePubMed
- Lun X, Alain T, Zemp FJ, Zhou H, Rahman MM, Hamilton MG, McFadden G, Bell J, Senger DL, Forsyth PA: Myxoma virus virotherapy for glioma in immunocompetent animal models: optimizing administration routes and synergy with rapamycin. Cancer Res. 2010, 70 (2): 598-608.View ArticlePubMed
- Boya P, Gonzalez-Polo RA, Casares N, Perfettini JL, Dessen P, Larochette N, Metivier D, Meley D, Souquere S, Yoshimori T, Pierron G, Codogno P, Kroemer G: Inhibition of macroautophagy triggers apoptosis. Mol Cell Biol. 2005, 25 (3): 1025-1040.PubMed CentralView ArticlePubMed
- Amaravadi RK, Yu D, Lum JJ, Bui T, Christophorou MA, Evan GI, Thomas-Tikhonenko A, Thompson CB: Autophagy inhibition enhances therapy-induced apoptosis in a Myc-induced model of lymphoma. J Clin Invest. 2007, 117 (2): 326-336.PubMed CentralView ArticlePubMed
- Enzenmuller S, Gonzalez P, Debatin KM, Fulda S: Chloroquine overcomes resistance of lung carcinoma cells to the dual PI3K/mTOR inhibitor PI103 by lysosome-mediated apoptosis. Anticancer Drugs. 2013, 24 (1): 14-19.View ArticlePubMed
- Ji C, Zhang L, Cheng Y, Patel R, Wu H, Zhang Y, Wang M, Ji S, Belani CP, Yang JM, Ren X: Induction of autophagy contributes to crizotinib resistance in ALK-positive lung cancer. Cancer Biol Ther. 2014, 15 (5): 570-577.PubMed CentralView ArticlePubMed
- Waqar SN, Gopalan PK, Williams K, Devarakonda S, Govindan R: A phase I trial of sunitinib and rapamycin in patients with advanced non-small cell lung cancer. Chemotherapy. 2013, 59 (1): 8-13.View ArticlePubMed
- Chaabane W, User SD, El-Gazzah M, Jaksik R, Sajjadi E, Rzeszowska-Wolny J, Los MJ: Autophagy, apoptosis, mitoptosis and necrosis: interdependence between those pathways and effects on cancer. Arch Immunol Ther Exp (Warsz). 2013, 61 (1): 43-58.View Article
- Jain MV, Paczulla AM, Klonisch T, Dimgba FN, Rao SB, Roberg K, Schweizer F, Lengerke C, Davoodpour P, Palicharla VR, Maddika S, Los M: Interconnections between apoptotic, autophagic and necrotic pathways: implications for cancer therapy development. J Cell Mol Med. 2013, 17 (1): 12-29.PubMed CentralView ArticlePubMed
- Alain T, Lun X, Martineau Y, Sean P, Pulendran B, Petroulakis E, Zemp FJ, Lemay CG, Roy D, Bell JC, Thomas G, Kozma SC, Forsyth PA, Costa-Mattioli M, Sonenberg N: Vesicular stomatitis virus oncolysis is potentiated by impairing mTORC1-dependent type I IFN production. Proc Natl Acad Sci U S A. 2010, 107 (4): 1576-1581.PubMed CentralView ArticlePubMed
- Thomas DL, Doty R, Tosic V, Liu J, Kranz DM, McFadden G, Macneill AL, Roy EJ: Myxoma virus combined with rapamycin treatment enhances adoptive T cell therapy for murine melanoma brain tumors. Cancer Immunol Immunother. 2011, 60 (10): 1461-1472.PubMed CentralView ArticlePubMed
- Yu H, Su J, Xu Y, Kang J, Li H, Zhang L, Yi H, Xiang X, Liu F, Sun L: p62/SQSTM1 involved in cisplatin resistance in human ovarian cancer cells by clearing ubiquitinated proteins. Eur J Cancer. 2011, 47 (10): 1585-1594.View ArticlePubMed
- Ajabnoor GM, Crook T, Coley HM: Paclitaxel resistance is associated with switch from apoptotic to autophagic cell death in MCF-7 breast cancer cells. Cell Death Dis. 2012, 3: e260-PubMed CentralView ArticlePubMed
- Veldhoen RA, Banman SL, Hemmerling DR, Odsen R, Simmen T, Simmonds AJ, Underhill DA, Goping IS: The chemotherapeutic agent paclitaxel inhibits autophagy through two distinct mechanisms that regulate apoptosis. Oncogene. 2013, 32 (6): 736-746.View ArticlePubMed
- Bertolini G, Roz L, Perego P, Tortoreto M, Fontanella E, Gatti L, Pratesi G, Fabbri A, Andriani F, Tinelli S, Roz E, Caserini R, Lo Vullo S, Camerini T, Mariani L, Delia D, Calabro E, Pastorino U, Sozzi G: Highly tumorigenic lung cancer CD133+ cells display stem-like features and are spared by cisplatin treatment. Proc Natl Acad Sci U S A. 2009, 106 (38): 16281-16286.PubMed CentralView ArticlePubMed
- Barr MP, Gray SG, Hoffmann AC, Hilger RA, Thomale J, O'Flaherty JD, Fennell DA, Richard D, O'Leary JJ, O'Byrne KJ: Generation and characterisation of cisplatin-resistant non-small cell lung cancer cell lines displaying a stem-like signature. PLoS One. 2013, 8 (1): e54193-PubMed CentralView ArticlePubMed
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/14/551/prepub
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.